PP026-MON HEPCIDIN STORM AFTER ABDOMINAL SURGERY

Abstract

Rationale: Glucagon-like peptide-1 (GLP-1) and its potent agonists have been widely studied in pancreatic islet b-cells. However, GLP-1 receptors are present in many extrapancreatic tissues including macrophages, and thus GLP-1 may have diverse actions on these tissues and cells. Therefore, we examined the mechanism by which exendin-4 (EX-4), a potent GLP-1 receptor agonist, inhibits lipopolysaccharide (LPS)-induced iNOS expression in Raw264.7 macrophage cells. Methods: The expression of iNOS, p-IuBa and p65 were detected by Western blot analysis and content of nitrite was measured using Griess reagent. Additionally, iNOS mRNA expression and promoter activity were observed by Northern blot analysis and luciferase assay, respectively. To observe the stability of iNOS mRNA and protein, actinomycin D chase and cycloheximide chase studies were performed, respectively. Also, we examined using adenylate cyclase inhibitor, PKA inhibitor and PKA gene silencing. Results: EX-4 significantly inhibited LPS-induced iNOS protein expression and nitrite production. However, EX-4 did not inhibit LPS-induced iNOS mRNA expression and iNOS promoter activity. Consistent with the result of iNOS promoter, LPS-induced IuBa phosphorylation and nuclear translocation of p65 were not inhibited by EX-4. Also, actinomycin D chase study showed that EX-4 did not affect iNOS mRNA stability. Meanwhile, cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. The EX-4 inhibition of LPS-induced iNOS protein was significantly reversed by adenylate cyclase inhibitors (MDL-12330A and SQ 22536), a PKA inhibitor (H-89) and PKAa gene silencing. Conclusion: These findings suggest that EX-4 inhibited LPS-induced iNOS protein expression at protein level, but not at transcriptional mechanism of iNOS gene and this inhibitory effect of EX-4 was mainly dependent on cAMP/PKA system.